Edwin J. Routledge

Structural Features of Alkylphenolic Chemicals Associated with Estrogenic Activity

The ability of certain man-made chemicals to mimic the effects of natural steroid hormones and their potential to disrupt the delicate balance of the endocrine system in animals are of increasing concern. The growing list of reported hormone-mimics includes the alkylphenolic (AP) compounds, a small number of which have been reported to be weakly estrogenic. In their most basic form, APs are composed of an alkyl group, which can vary in size, branching, and position, joined to a phenolic ring. The aim of this project was to identify the important structural features responsible for the estrogenic activity of AP chemicals. This was achieved by incubating APs with different structural features in a medium containing a previously described estrogen-inducible strain of yeast (Saccharomyces cerevisiae) expressing the human estrogen receptor and comparing their activity spectrophotometrically by the resulting color change of the medium. The results were compared to the effects of the main natural estrogen 17β-estradiol. The data indicate that both the position (para > meta > ortho) and branching (tertiary > secondary = normal) of the alkyl group affect estrogenicity. Optimal estrogenic activity requires a single tertiary branched alkyl group composed of between 6 and 8 carbons located at the para position on an otherwise unhindered phenol ring. The results are discussed in relation to the purity and composition of the chemicals tested.

Benzotriazole is antiestrogenic in vitro but not in vivo

Benzotriazole (BT) is an anticorrosive agent well known for its use in aircraft deicing and antifreeze fluids but also used in dishwasher detergents. It is highly persistent in the environment; therefore, BT is frequently found in runoff emanating from large airports as well as in the surrounding groundwater. In addition, BT has recently been found to be ubiquitous in Swiss wastewater treatment plant effluents and their receiving waters; however, very little chronic toxicity data is available on which to base a sound ecological risk assessment of this chemical. In vitro assays conducted using a recombinant yeast (anti-) estrogen assay indicated that BT possessed clear antiestrogenic properties. This chemical was approximately 100-fold less potent than Tamoxifen, which was used as a positive control. A subsequent in vivo study, however, involving analysis of vitellogenin induction and somatic indices in adult fathead minnows (Pimephales promelas) exposed to BT at concentrations of 10, 100, and 1,000 mug/L for two weeks showed no evidence of antiestrogenic activity by this compound. The possibility exists that higher concentrations of BT may yet induce the type of activity observed in vitro, although the concentrations used here already far exceed those reported in surface-water samples. Furthermore, adverse effects may be observed in fish or other organisms exposed to BT for a longer period than employed here, although such studies are costly and unlikely to be included in standard risk assessment procedures. A rigorous investigation of the chronic toxicity of BT is imperative.

17β-Oestradiol may prolong reproduction in seasonally breeding freshwater gastropod molluscs.

Whilst the effects of oestrogenic contaminants in the aquatic environment are well documented in fish, effects in invertebrate species has been subject to debate, possibly due to differences in experimental conditions (temperature, timing and duration of exposure) between studies. It has been suggested that molluscs are only susceptible to oestrogens in periods either following the main spawning or leading up to the maturation of gametes. To investigate this possibility, two temperate, seasonally reproducing gastropods (Planorbarius corneus and Viviparus viviparus) were exposed to two concentrations of 17β-oestradiol (E2; 10ng/l and 100ng/l nominal) in an outdoor mesocosm (subject to natural seasons). In addition, P. corneus was also exposed to E2 (1, 10 and 100ng/l) in the laboratory at temperatures and photoperiods to simulate summer and autumn. In the mesocosm, both snail species produced similar numbers of eggs/embryos as reference groups in the summer, but the groups exposed to 10ngE2/l (nominal) had significantly higher productivities after the onset of autumn, when entering their quiescent phase, whilst the snails exposed to a higher concentration (100ng/l, nominal) had an increased rate of mortality, and did not experience increased reproduction. In the laboratory, the rate of egg laying in P. corneus was unaffected in simulated summer (20°C, 16h photoperiod), but snails exposed to 10 and 100ng/l (nominal) in simulated autumn (15°C, 12h photoperiod) showed a concentration-dependent inhibition of the natural decline in egg laying observed in the control snails. Overall, rather than an increase in reproductive rate, the response of this species was a perpetuation of summer reproductive rates into autumn. We conclude that exposure to E2 can affect reproduction in the freshwater gastropods studied, but in P. corneus at least, this is dependent on the seasonal conditions (temperature and photoperiod) at which exposures are made.

The Nuclear Receptors of Biomphalaria glabrata and Lottia gigantea: Implications for Developing New Model Organisms

Nuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snails potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.

Agricultural intensity in ovo affects growth, metamorphic development and sexual differentiation in the Common toad (Bufo bufo)

Pollution was cited by the Global Amphibian Assessment to be the second most important cause of amphibian decline worldwide, however, the effects of the agricultural environment on amphibians are not well understood. In this study, spawn from Bufo bufo was taken from four sites in England and Wales with varying intensities of arable agriculture. Spawn was either placed in tanks containing aged tap water (ex-situ, five replicates) or in cages at the native site (caged, five replicates). Hatching success, abnormal tadpoles, and forelimb emergence were recorded during the larval stage. Individuals were also sampled at five time points (TP) during development (5-, 7-, 9-, 12-, 15-weeks post-hatch) and analysed for morphological parameters. The thyroids (TP2) and the gonads (TP3,4,5) were also analysed histologically. With the exception of the thyroid histopathology, all analysed endpoints were significantly different between ex-situ individuals reared under identical conditions from the different sites. In addition, intensity of arable agriculture had a negative effect on growth and development. At one site, despite distinct rearing conditions, a high level of intersex (up to 42%) and similar sex ratios were observed in both ex-situ and caged individuals. Taken together, these data suggest that maternal exposure and/or events in ovo had a much larger effect on growth, metamorphic development, and sexual differentiation in B. bufo than the ambient environment. This could have important implications for traditional exposure scenarios that typically begin at the larval stage. Intersex is reported for the first time in European amphibians in situ, highlighting the potential use of distinct populations of amphibians in fundamental research into the aetiology of specific developmental effects in wild amphibians.

Plants Used in Chinese Medicine for the Treatment of Male Infertility Possess Antioxidant and Anti-Oestrogenic Activity

In this study Chinese herbs commonly used in the treatment of male infertility were investigated for relevant biochemical activity. Male factor infertility predominantly arises via barriers to, or defects in, spermatogenesis. The process of spermatogenesis is under strict endocrine control; in addition oxidative stress has been implicated in male infertility with significant levels of reactive oxygen species detected in 25% of infertile males. A total of 37 individual herbs and seven herb decoctions used in the treatment of male factor infertility were therefore tested for endocrine activity using a recombinant yeast based assay and antioxidant activity using the FRAP (ferric reducing antioxidant potential) assay. Individual herbs tested did not show androgenic properties, 20 showed strong and 10 weak anti-oestrogenic activity (per g of dried herb tamoxifen equivalents ranged from 1.18–1280.66 mg and 0.06–0.98 mg, respectively). Oestrogenic responses were elicited for two herbs (85.30–550 μg oestradiol equivalents/g dried herb), with seven and three herbs exhibiting a strong or weak anti-androgenic response (per g of dried herb DHT equivalents ranged from 1.54–66.78 mg and 0.17–0.32 mg), respectively. Of these 37 herbs, strong (15 herbs), intermediate (7 herbs) and weak/no (15 herbs) antioxidant activity was detected (ranging from 0.912–1.26; 0.6–0.88 and 0–0.468 μg ascorbate equivalent/mg dried herb, respectively). The seven decoctions (previously used to treat patients) tested elicited strong (5 herbs) and weak (2 herbs) anti-oestrogenic responses (per g of dried herb tamoxifen equivalents ranged from 1.14–13.23 mg and 0.22–0.26 mg, respectively), but not oestrogenic, androgenic nor anti-androgenic, consistent with their individual composition. With regard to antioxidant activity the following responses were recorded: three strong, three intermediate and one weak (ranging from 1.02–1.2; 0.72–0.76 and 0.44 μg ascorbate equivalent/mg dried herb, respectively). The prospects for introducing Chinese herbal treatments into the Western-based medicine are discussed.

No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals

Highlights • ER and ERR transcription levels were unaffected in Marisa cornuarietis exposed to 17β-estradiol or 4-tert-Octylphenol.• The mollusc ER protein interacts with the phytoestrogen genistein in transfected HEK-293 cells.• The mollusc ERR protein interacts weakly with bisphenol-A in transfected HEK-293 cells.• The mollusc ER protein binds to the vertebrate consensus estrogen response element (ERE) sequence.

Impact of an environmentally-realistic intake of cadmium on calcium, magnesium, and phosphate metabolism in bank voles, Clethrionomys glareolus

Oral intake of cadmium can disrupt calcium metabolism in laboratory rodents. If this occurs in free-living wild rodents, reproductive potential, growth, and survival could be adversely affected. This study investigated whether an environmentally-realistic intake of cadmium (1.5–1.7 mg/kg BW/24h) by bank voles, Clethrionomys glareolus, affected uptake and assimilation of calcium and also that of magnesium and phosphorus, two elements metabolized in close association with calcium. Mineral metabolism was monitored in bank voles fed four different diets; two diets were relatively low (0.12%) in calcium and contained either no cadmium (LC) or cadmium (LCCd), while the remainder were relatively high (0.74%) in calcium and also contained either no cadmium (HC) or cadmium (HCCd). Dietary calcium levels were varied because calcium can affect gut absorption of cadmium, and calcium levels in natural forage vary with habitat soil type.Cadmium had a significant effect on voles fed a low calcium diet. Animals given LCCd diet had significantly poorer calcium net gut absorption efficiency than animals fed the corresponding control (LC) diet and were in negative calcium balance, losing approximately 0.5–1% of body calcium reserves each day. In contrast, calcium inputs matched total outputs in animals fed the LC diet. Cadmium had no effect on net gut absorption of magnesium and phosphorus, but renal reabsorption and subsequent assimilation of these elements was greater in animals fed the LCCd diet than in those given the LC diet.Cadmium had no clear effect on macroelement metabolism in bank voles fed a diet relatively high in calcium. Net gut absorption and assimilation of calcium by voles fed the HCCd diet was reduced to the same extent as observed in animals fed the LCCd diet. However, because of their high dietary calcium intake, voles fed the HCCd diet maintained a positive calcium balance and there was no significant difference in calcium assimilation between animals fed the HCCd and HC diet. Cadmium had no effect on magnesium and phosphorus metabolism in voles fed high calcium diets.

Low-cost motility tracking system (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation.

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17β-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17β-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.